Figure 1. Movies of Sec61-ß in ER. Top left: Z-stack of live hiPS cells expressing mEGFP tagged Sec61-ß imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Top right: Timelapse movie of live hiPS cells expressing mEGFP tagged Sec61-ß. Images were collected in 3D every 2 minutes for 3 hours on a spinning-disk confocal microscope. Image is a single slice through the center of the cells. Playback speed is 600x real time. Bottom image panel: live hiPSC cells expressing mEGFP tagged Sec61-ß imaged on a Zeiss LSM 880 AiryScan FAST in super-resolution mode.

Figure 2. Images of Sec61-ß in ER. Left, middle, and right images represent a single slice at the bottom, center, and top of cells with AiryScanFast SuperRes

Observations

Sec61-ß is a member of the Sec61 complex, which is involved in protein translocation and insertion into the ER membrane.

In hiPS cells, the ER is localized to the nuclear periphery and in tubules and sheet-like structures throughout the cytoplasm.

ER morphology differs at the top vs. the bottom of cells. The ER is more densely packed near the top of cells such that the tubules have a highly branched appearance. The tubules appear longer and less branched at the bottom of cells. This pattern of increased organelle density near the top and decreased near the bottom of cells is similar to that of mitochondria and may also be due to apical-basal variation in microtubule positioning within cells.

During cell division, the ER stays mostly intact but the peripheral ER takes on a wavy morphology very similar to that of the nuclear envelope. After division, as the peripheral ER reforms, similar membrane invaginations are seen as in LaminB1 tagged cells. This suggests these invaginations might be composed of both nuclear envelope and ER. These invaginations disappear with time during interphase.